Nucleic acid detection using probes

Abstract

The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that complementary to the regulatory sequence and a tail segment that does not hybridize to the probe nucleotide when the sequence segment and the regulatory sequence are annealed, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and using a DNA polymerase with high strand displacement activity and low 5′-nuclease activity, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.

We claim: 
     
       1. A method for detecting a target nucleotide sequence comprising:
 a) tagging the target nucleotide sequence with a nucleotide tag sequence, thereby producing a tagged target nucleic acid sequence; 
 b) providing a probe oligonucleotide comprising a nucleotide tag recognition sequence complementary to the nucleotide tag sequence and a regulatory sequence 5′ to the nucleotide tag recognition sequence, wherein said probe oligonucleotide comprises a first label and has a melting temperature Tm1; 
 c) amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, wherein said PCR amplification reaction is characterized by an annealing temperature Ta; 
 wherein the PCR amplification reaction is carried out in the presence of a regulatory oligonucleotide comprising a sequence segment that is complementary to the regulatory sequence and a tail segment with a sequence not complementary to the probe oligonucleotide sequence, wherein said regulatory oligonucleotide comprises a second label and has a melting temperature Tm2; and 
 d) detecting the product of the PCR amplification reaction; 
 wherein the first label and the second label constitute a fluorescent reporter/quencher pair; and wherein Tm1 and Tm2 are both higher than Ta. 
 
     
     
       2. The method of  claim 1 , wherein the nucleotide tag sequence is incorporated into the tagged target nucleic acid sequence using a PCR reaction. 
     
     
       3. The method of  claim 1 , wherein the nucleotide tag recognition sequence is exactly complementary to the nucleotide tag sequence. 
     
     
       4. The method of  claim 1 , wherein the regulatory oligonucleotide comprises a sequence segment that is exactly complementary to the regulatory sequence. 
     
     
       5. The method of  claim 1 , wherein the regulatory oligonucleotide has a length in the range of 15-45 nucleotides. 
     
     
       6. The method of  claim 1 , wherein the Ta is in the range of 55-64° C. 
     
     
       7. The method of  claim 1 , wherein the PCR amplification reaction comprises at least 20 cycles at the Ta. 
     
     
       8. The method of  claim 1 , wherein the tail segment of the regulatory oligonucleotide has a length of from 5 to about 25 bases. 
     
     
       9. The method of  claim 8 , wherein the tail segment has a length of 5-8 bases. 
     
     
       10. The method of  claim 8 , wherein the tail segment is at least about 70% GC. 
     
     
       11. The method of  claim 1 , wherein the tail segment of the regulatory oligonucleotide is 100% noncomplementary to the region of the probe oligonucleotide to which it corresponds when the regulatory oligonucleotide is aligned to the probe oligonucleotide.